Throughout this application, various publications are referenced by Arabic numerals with parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein.
The term transforming growth factor (TGF) has been applied to peptides that have the ability to confer the transformed phenotype on untransformed fibroblastic indicator cells in vitro. In the early 1980's it was suggested that polypeptide growth factors secreted by a cell might play a part in malignant transformation of that cell. The demonstration that many virally transformed cells which had reduced levels of assayable receptors for epidermal growth factor (EGF), also secreted into their conditioned media substances which competed binding to EGF receptors. Support for this concept of "autocrine" secretion (1,2) was provided by the discovery of acid-stable, heat-stable peptides, called sarcoma growth factor in the conditioned media of Moloney Sarcoma virus transformed cells (3). Sarcoma growth factor not only had the property of binding to the EGF receptor but could also induce the reversible transformation of rat kidney fibroblasts (NRK). This anchorage independent growth in soft agar is now accepted as the operational definition of transforming growth factor (TGF).
Peptides representing two distinct classes of TGF'S have been purified to homogeneity. TGF.alpha. and TGF-.beta. are distinguished biochemically by their unique amino acid sequences and biologically by their different activities on cells. TGF.alpha. is a single polypeptide chain of 50 amino acids containing three disulfide bonds. TGF-.beta. is a homodimeric structure consisting of two chains of 112 amino acids, each containing nine cysteine residues. The ability to induce anchorage independent growth of NRK cells requires the combined action of TGF.alpha. and TGF-.beta. (4).
TGF.alpha. is a 6-kd protein which binds to the EGF receptor (5). TGF.alpha. shares 33% sequence homology with EGF and has an affinity for the EGF receptor similar to EGF (6). Derynck et al. (7) have shown that TGF.alpha. is synthesized as a larger precursor of 160 amino acids. The larger precursor protein is glycosylated and palmitoylated and is thought to be a transmembrane protein that sequentially undergoes external proteolytic cleavage, releasing TGF.alpha. species ranging in molecular weight from 6 kd to 25 kd (8).
Examination of a wide variety of human tumors by northern hybridization indicates that squamous, renal, mammary carcinomas as well as melanoma synthesize TGF.alpha. mRNA (9). TGF.alpha. has also been detected in the urine of cancer patients (10-13) but is also expressed in both fetal and normal tissues. In the mouse embryo TGF.alpha. mRNA expression peaks at day 9 and quickly levels off by day 21 (14). TGF.alpha. has been detected in human keratinocytes, in bovine anterior pituitary and bovine ovarian theca-interstitial cells (15-18). The function of TGF.alpha. in normal cells is presently not known.
It has been suggested that tumor and fetal derived EGF-like peptides are structurally related to TGF.alpha. while normal cells produce growth factors related to EGF(1,2). In many cases biological assays have been used to detect the presence of TGF.alpha.; however, because of their similarities, biological assays cannot be used to distinguish TGF.alpha. from EGF. Currently, there are a limited number of monoclonal antibodies which have been made against TGF.alpha. (8,18,19). We have also produced a series of monoclonal antibodies specific for TGF.alpha. which do not crossreact with EGF. We have used these antibodies to: specifically detect TGF.alpha. in conditioned media of tumor cells; 2) detect TGF.alpha. in the urine of cancer patients using a TGF.alpha. immunometric assay and by immunoblotting; 3) and to identify TGF.alpha. in tumor cells and paraffin-embedded tissues by immunohistological staining.